Screening And Identification Of Radiosensitivity-related Serological MicroRNAs In Nasopharyngeal Carcinoma

Part One SCREENING OF RADIOSENSITIVITY-RELATED SEROLOGICAL MICRORNAS IN NASOPHARYNGEAL CARCINOMABackground and objective:Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in Guangxi Zhuang Autonomous Region.Due to the specific anatomic site and characteristic of radiosensitivity,integrated treatment pattern which radiotherapy contributed mostly was still a prevalent means at present.In recent years,with the development of radiotherapy equipments,radiotherapy technology,radiophysics and radiobiology,the therapeutic effect of nasopharyngeal carcinoma has been further improved.However,NPC patients with the similar characters of pathologic category,clinical stage,age and status treated by the same radiotherapy technology and dose had so different curative effects in clinic.Some patients even recurred in the irradiation field.The main reason for this condition is radioresisitant.Therefore,it is particularly urgent to accurately predict the radiosensitivity of nasopharyngeal carcinoma patients before radiotherapy and to formulate more reasonable individualized radiotherapy programs for patients.On the basis of previous studies on the mechanism of radioresistance of NPC cell lines,our team intend to screen the specific microRNAs related to radiosensitivity of NPC patients by miRNA array technology,and construct the differential expression profiles of radioresistance-related miRNAs in NPC,so as to find out the biomarkers that can be used to evaluate the radiosensitivity of NPC in clinic and elucidate the molecular mechanism of radioresistance in NPC.This study will be of significance in guiding the prediction of radiosensitivity,and it will be useful to the conduct individualized treatment of NPC.Method:(1)We collected the serum samples of NPC patients of different radiosensitivity,including 7 radiosensitive NPC patients and 5 radioresistant NPC patients.All patients were strictly enrolled according to the standard of inclusion criteria.Blood collection was carried out before any treatment was accepted.After blood collection,serum was collected by centrifugation.After packaging,the serum was labeled and stored in the refrigerator at-80?.(2)Then we performed the microarray test.RNA of the NPC patients as samples for array test was extracted.The expression levels of miRNAs in two groups were detected and normalized.The differentially expressed miRNAs between the two groups were screened.(3)The microWalk 2.0 online software was used to predict target genes of differential miRNAs.ClusterProfiler package in R was used to perform GO analysis and KEGG pathway annotation enrichment analysis to predict the functional classification and signaling pathway of differential genes.Results:(1)We screened out of 37 miRNAs with significant differential expression(Fold change>2,P value<0.05)between the radiosensitive NPC patients and the radioresistant NPC patients by microarray test.Compared with the radiosensitive group,19 miRNAs were up-regulated and 18 miRNAs were down-regulated in the radioresistant group.(2)MiRWalk 2.0 online software was used to predict target genes of the differential miRNAs.It showed the predicted target genes of 12 prediction programs.We collected 8824 target genes which were screened out from at least 6 programs.(3)Bioinformatic analysis of predicted target genes of differentially expressed miRNAs showed that Ras protein signal transduction?axonogenesis?axon development were the main biological processes involved.Cellular components were mainly enriched in cell leading edge?Postsynapse and postsynaptic specialization.Molecular functions were mainly enriched in DNA binding.And the three most significant enriched pathways were MAPK signaling pathway,Axon guidance,Proteoglycans in cancer.The most significant enriched diseases were malignant glioma,organ system benign neoplasm and connective tissue cancer.Conclusion:In this study,37 serum-specific miRNAs were screened out from NPC patients of different radiosensitivity by microarray test,and the differential expression profiles of miRNAs in serum from radioresistant NPC patients were preliminarily constructed.Part Two IDENTIFICATION OF RADIOSENSITIVITY-RELATED SEROLOGICAL MICRORNAS IN NASOPHARYNGEAL CARCINOMABackground and objective:In the previous study,37 miRNAs related to radiosensitivity of NPC were screened by microarray test.Comparing to the radiosensitive group,19 miRNAs were up-regulated and 18 miRNAs were down-regulated in the radioresistant group.In order to further verify the reliability of microarray results,we selected some significantly differential expression miRNAs for reverse transcription-quantitative real time polymerase chain reaction(RT-qPCR)experiments to evaluate the feasibility of serum miRNAs as biomarkers for predicting radiosensitivity of NPC,so as to provide theoretical basis for further exploring the molecular mechanism of NPC radioresistance and performing the individualized diagnosis and treatment of NPC.Method:Serum samples from newly diagnosed NPC patients with different radiosensitivity were collected before treatment.RT-qPCR was used to verify the different expressions of hsa-miR-1281,hsa-miR-1825,hsa-miR-6732-3p and hsa-miR-6865-3p,which screened by microarray test.In order to test the predictive value of differentially expressed miRNAs for estimating the radiosensitivity of NPC,the ROC(Receiver Operating Characteristic)curve and AUC(Area under the Curve of ROC)were further used for analysis in this study.Results:Through RT-qPCR test,we found that the relative expression levels of hsa-miR-1281 and hsa-miR-6732-3p in serum of the radioresistant NPC patients were significantly lower than those of the radiosensitive NPC patients(P<0.05),which was consistent with the results of microarray test.The AUC of hsa-miR-1281 was 0.737(95%Cl:0.556-0.917,P=0.028),and the sensitivity and specificity were 87.5%and 57.1%,respectively.While the AUC of hsa-miR-6732-3p was 0.692(95%Cl:0.501-0.883,P=0.074),and the sensitivity and specificity were 56.3%and 85.7%,respectively.The diagnostic accuracy of hsa-miR-1281 and hsa-miR-6732-3p in combination for NPC(AUC=0.741,95%Cl:0.561-0.921,P=0.025)was higher than that of hsa-miR-1281 or hsa-miR-6732-3p alone.Conclusion:(1)RT-qPCR test showed that hsa-miR-1281 had moderate diagnostic ability and certain accuracy in estimating the radiosensitivity of NPC patients,while hsa-miR-6732-3p had lower diagnostic ability.(2)Because of the convenience of serum sample collection and the relative noninvasiveness,it is valuable of hsa-miR-1281 and hsa-miR-6732-3p in serum to evaluate the radiosensitivity of NPC patients as biomarkers.But its reliability needs to be further validated in a larger sample population.